The ability to coordinate and control cell-cell interactions is a pre-requisite for the evolution and development of multicellular life forms. Tissues and organs, such as the central nervous system and immune system, for example, are composed of multiple cellular types. The functioning of these systems is highly dependent on the expression of cell membrane ligands and receptors that direct the interaction between cells. The development of approaches for the controlled display of ligands and receptors on cell surfaces would greatly facilitate the ability to manipulate the interactions of cells with other cells and tissues. In addition, these methodologies may also have potential also be used to track the behavior of cells in vivo by labeling cells with either optical, radiological or MRI detectable probes.
A number of approaches have been devised for the re-engineering of cell surfaces (Stephan, M. T. I., D. J. Enhancing Cell Therapies from the Outside In: Cell Surface Engineering using Synthetic Nanomaterials. Nano Today 6, 309-325, 2011). Certain methods have taken advantage of the promiscuity of glycan biosynthesis to randomly terminate cell surface glycoproteins with azido-neuramimic acid, followed by conjugation by click chemistry to oligonucleotides (Chandra, R. A., et al. Programmable cell adhesion encoded by DNA hybridization. Angewandte Chemie-International Edition 45, 896-901, doi:10.1002/anie.200502421, 2006), thus enabling control over cell-cell interactions by differential conjugation to complementary oligonucleotides (David Rabuka, et al. Site-specific Chemical Protein Conjugation using Genetically Encoded Aldehyde Tags. Nature Protocol 7, 1052-1066, 2012). Chemical cross-linking methods have also been developed that rely on the random biotinylation of cell surface macromolecules, followed by tethering to a ligand through avidin binding (Cheng, H. et al. Nanoparticulate Cellular Patches for Cell-Mediated Tumoritropic Delivery. ACS Nano 4, 625-631, doi:10.1021/nn901319y, 2010). The lack of specificity of these approaches may lead to unforeseen disruptions in either intracellular and extracellular glycoprotein biosynthesis or cell membrane function. In addition, while covalent modifications are highly stable, depending on the turnover of the membrane protein or oligosaccharide, they are not reversible.
Non-covalent cell surface modification approaches have received far less attention. Membrane intercalating proteins or peptides have been conjugated to a phospholipid or fatty acids or linked recombinantly to glycosylphosphatidylinositol (GPI) (Ko, I. K., et al. Targeting mesenchymal stem cells to activated endothelial cells. Biomaterials 30, 3702-3710, doi:10.10161j.biomaterials.2009.03.038, 2009). Chemically reactive fatty acids have been incorporated into liposomes and when allowed to fuse to cell membranes provide chemical conjugation sites to cellular membranes (Dutta, D., et al. Engineering Cell Surfaces via Liposome Fusion. Bioconjugate Chemistry 22, 2423-2433, doi:10.1021/bc200236m, 2011; Dutta, D., et al. Synthetic Chemoselective Rewiring of Cell Surfaces: Generation of Three-Dimensional Tissue Structures. Journal of the American Chemical Society 133, 8704-8713, doi:10.1021/ja2022569, 2011). Although, reactive fatty acids have been shown to distribute to the membranes of other cellular organelles, the ability to control cell adhesion to surfaces, including other cells, has been demonstrated (Dutta, D., et al. Bioconjugate Chemistry 22, 2423-2433, doi:10.1021/bc200236m, 2011; Dutta, D., et al. Journal of the American Chemical Society 133, 8704-8713, doi:10.1021/ja2022569, 2011). Unfortunately, although membrane protein function may not be directly affected, the association half-life of proteins conjugated to fatty acids or phospholipids is relatively short, ranging from one to two hours (Ko, I. K., et al. Targeting mesenchymal stem cells to activated endothelial cells. Biomaterials 30, 3702-3710, doi:10.1016/j.biomaterials. 2009.03.038, 2009; de Kruif, J., et al. Recombinant lipid-tagged antibody fragments as functional cell-surface receptors. Nat. Med. 6, 223-227, doi:10.1038/72339, 2000). Non-cytotoxic polymers have been used to coat cells, while methods have been developed for adhering polyelectrolyte multilayer (PEM) patches to cells using photolithography (Wilson, J. T., et al. Layer-by-layer assembly of a conformal nanothin PEG coating for intraportal islet transplantation. Nano Letters 8, 1940-1948, doi:10.1021/n1080694q, 2008; Swiston, A. J. et al. Surface Functionalization of Living Cells with Multilayer Patches. Nano Letters 8, 4446-4453, doi:10.1021/n1802404h, 2008). While the stability of the association of either polymer or electrolytes to cells is impressive, the modifications are irreversible and, particularly for photolithography, the number of cells that can be modified is limiting. Other surface modification methods are those that rely on molecular biological techniques to genetically engineer cells to express receptors or ligands. For example, T-cells of Adult Lymphocytic Leukemia (ALL) patients have been engineered to express an anti-CD 19 single-chain antibody fused to CD3ε, referred to as chimeric antigen receptors (CARs) (Porter, D. L., et al. Chimeric Antigen Receptor-Modified T cells in chronic Lymphoid Leukemia. The New England Journal of Medicine 365, 725-734, 2011). The engineered CARS T-cells have demonstrated the ability to suppress B-lymphocytic tumor growth clinically (Brentjens, R. J. et al. CD19-Targeted T Cells Rapidly Induce Molecular Remissions in Adults with Chemotherapy-Refractory Acute Lymphoblastic Leukemia. Science Translational Medicine 5, doi:10.1126/scitranslmed.3005930, 2013; Porter, D. L. et al Chimeric Antigen Receptor T Cells Directed Against CD19 Induce Durable Responses and Transient Cytokine Release Syndrome in Relapsed, Refractory CLL and ALL. Blood 120, 2012). Nevertheless, the relatively low transfection efficiency of these methods and the unknown clinical outcome of long lived genetically modified cells are challenges that remain to be addressed (Curran, K., et al. Chimeric Antigen Receptors for T cell Immunotherapy: Current Understanding and future directions. The Journal of Gene Medicine 14, 405-415, 2012). Consequently, a non-genetic approach that would allow the rapid, stable and reversible modification of membranes with one or more ligands would provide a complementary synthetic biological tool for the re-programming of cell surfaces.
A method for the engineering and preparation of chemically self-assembled nanorings (CSANs) has been developed (Carlson, J. C. T. et al. Chemically controlled self-assembly of protein nanorings. J. Amer. Chem. Soc. 128, 7630-7638, 2006). CSANs are prepared by taking advantage of the power of high affinity chemically-induced dimerization (Fegan, A., et al. Chemically controlled protein assembly: Techniques and applications. Chem. Rev. 110, 3315-3336, 2010). When mixed with a covalently linked dimer of the dihydrofolate reductase (DHFR) inhibitor methotrexate (bisMTX), DHFR forms highly robust protein dimers with an affinity of approximately 10−11 M (Carlson, J. J. Amer. Chem. Soc. 128, 7630-7638, 2006). When one DHFR is recombinantly fused through an encoded linker peptide to another DHFR (yielding DHFR-DHFR or DHFR2), spontaneous and rapid self-assembly into CSANs was observed, whose diameter is dependent on the length and composition of the linker peptide (13-amino acid linker=dimer, 1-amino acid linker=octamer) (Li, Q., et al. Self-Assembly of Antibodies by Chemical Induction. Angewandte Chemie-International Edition 47, 10179-10182, doi:10.1002/anie.200803507, 2008); Li, Q. et al. Chemically Self-Assembled Antibody Nanorings (CSANs): Design and Characterization of an Anti-CD3 IgM Biomimetic. J. Amer. Chem. Soc. 132, 17247-17257, doi:10.1021/ja107153a, 2010). The rings exhibit high stability with Tms ranging from 63-66° C. Single molecule experiments have also confirmed that even at picomolar concentrations, nearly 70% of the nanorings remain intact. Since the CSANs exhibit the properties of a stable scaffold, CSANs recombinantly fused to single-chain antibodies (scFvs) and peptides that target cell surface receptors were prepared (Li, Q., et al. Angewandte Chemie-International Edition 47, 10179-10182, doi:10.1002/anie.200803507, 2008; Li, Q. et al. Biomimetic. J. Amer. Chem. Soc. 132, 17247-17257, doi:10.1021/ja107153a, 2010; Gangar, A. et al. Targeted delivery of antisense oigonucleotides by chemically self-assembled nanostructures (CSANs). Mol. Pharmaceutics (ASAP), 2013). The resulting monovalent, bivalent or octavalent targeted-CSANs were found to selectively bind targeted cellular receptors. For example, octavalent anti-CD3 CSANs were shown to bind CD3+ lymphocytic cells with an affinity of 0.9 nM (Li, Q., et al. Angewandte Chemie-International Edition 47, 10179-10182, doi:10.1002/anie.200803507, 2008; Li, Q. et al. Biomimetic. J. Amer. Chem. Soc. 132, 17247-17257, doi:10.1021/ja107153a, 2010), while CSANs displaying the cyclic-RGD peptide were shown to target αvβ3 on breast cancer cells (Gangar, A. et al. Mol. Pharmaceutics (ASAP), 2013). Recently, the utility of CSANs has been expanded through the design and preparation of new bisMTX chemical dimerizers that contain a third arm with a reactive group capable of being conjugated to fluorophores, drugs and oligonucleotides (Gangar, A. et al. Mol. Pharmaceutics (ASAP), 2013; Fegan, A., et al. Chemically self-assembled antibody nanostructures as potential drug carriers. Mol. Pharmaceutics 9, 3218-3227, 2012; Gangar, A., et al. Programmable Self-Assembly of Antibody-Oligonucleotide Conjugates as Small Molecule and Protein Carriers. J. Amer. Chem. Soc. 134, 2895-2897, doi:10.1021/ja210894g, 2012). In addition, it has been demonstrated that CSANs can undergo rapid disassembly, both extra- and intracellularly, in the presence of clinically relevant doses of the non-toxic FDA approved bacterial DHFR inhibitor, trimethoprim; thus affording pharmacological and therefore temporal control of their interactions with cells and tissues(Li, Q., et al. Angewandte Chemie-International Edition 47, 10179-10182, doi:10.1002/anie.200803507, 2008; Li, Q. et al. Biomimetic. J. Amer. Chem. Soc. 132, 17247-17257, doi:10.1021/ja107153a, 2010; Fegan, A., Kumarapperuma, S. C. & Wagner, C. R. Chemically self-assembled antibody nanostructures as potential drug carriers. Mol. Pharmaceutics 9, 3218-3227, 2012). Accordingly, there is a need to develop agents including CSANs for the modification or re-engineering of cell surfaces for such uses as interrogation of cellular interactions in vitro and/or in vivo or the design of cell or tissue-based therapies such as the treatment of diseases (e.g., cancer).